Extraction of genomic DNA from a single adult schistosome

Extraction of genomic DNA from a single adult schistosome

Homogenization Buffer: 100 mM NaCl, 200 mM sucrose, 10 mM EDTA, 30 mM Tris pH 8.0
Lysis Buffer: 250 mM EDTA, pH 8.0, 2.5% SDS, 500 mM Tris pH 9.2
Grinding Buffer: 80% Homogenization Buffer, 20 % Lysis Buffer
  1. Grind one (to five) adult worm in 25 μl Grinding Buffer in 1.5. ml tube. Rinse the pestle with 25 μl fresh Grinding Buffer, and add the wash to the sample.
  2. 65 °C, 30 minutes
  3. Add 8 M ammonium acetate to a final concentration of 1 M (i.e. ~ 7 μl)
  4. Incubate on wet ice for 30 to 60 minutes
  5. Spin in Eppendorf centrifuge for 10 minutes. Transfer supernatant to a new 1.5 ml tube.
  6. Add 100 μl 100% ethanol; mix; and incubate at room temperature 5 minutes.
  7. Microfuge for 15 minutes (preferably at 4°C), remove ethanol carefully
  8. Wash barely visible pellets with 100% ethanol once, remove ethanol carefully, let tube air dry but not over dry. Add ~ 30 μl TE, pH 8.0 (10 mM Tris, 1 mM EDTA)
  9. Analyze by restriction digestion, spectrophotometry, PCR or other suitable methods.